Group a streptococcal vaccines

ABSTRACT

The present invention provides methods for eliciting an immune response against Group A streptococci, comprising use of recombinant fusion polypeptides, and compositions thereof, that include a multivalent immunogenic portion of at least two immunogenic polypeptides from Group A streptococci M proteins (which are capable of stimulating a protective immune response against Group A streptococci), and a reiterated polypeptide from the immunogenic portion carboxy-terminal to the immunogenic portion, wherein the carboxy-terminal polypeptide is not required to stimulate an immune response against Group A streptococci.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.09/151,409, filed Sep. 10, 1998, which application claims the benefit ofU.S. Provisional Application No. 60/058,635, filed Sep. 12, 1997, whichapplications are incorporated by reference in their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under Contract No. AI10085-33 awarded by the National Institutes of Health and Grant No.614-001 awarded by the Veteran's Administration. The government may havecertain rights in this invention.

TECHNICAL FIELD

The present invention provides pharmaceutical compositions and methods,and in particular, vaccines for use in preventing Group A streptococcalinfections.

BACKGROUND OF THE INVENTION

Streptococci are a group of bacteria with the capacity to grow inchains. Many varieties are part of the normal bacterial flora in humansand are not especially harmful. However, a particular group ofstreptococcal bacteria, called group A and represented by Streptococcuspyogenes, is a human pathogen. Briefly, group A streptococci cause avariety of human illnesses, ranging from uncomplicated pharyngitis andpyoderma to life-threatening infections associated with toxic shocksyndrome, deep tissue invasion and sepsis. In some individuals,untreated streptococcal pharyngitis may be followed by acute rheumaticfever. In recent years there has been a dramatic increase in theincidence of severe streptococcal infections (Davies et al., “Invasivegroup A streptococcal infections in Ontario, Canada. Ontario group AStreptococcal study group,” N. Engl. J. Med 335: 547-554, 1996) and inthe incidence of rheumatic fever (Veasey et al., “Resurgence of acuterheumatic fever in the intermountain region of the United States,” N.Eng. J. Med. 316: 421-427, 1987).

Although streptococcal infections can be generally treated withantibiotics, in at least 4% of cases the infection leads to acuterheumatic fever. This disease is particularly prevalent in developingcountries such as India, where millions of school-age children areaffected.

The present invention provides new Group A streptococcal vaccines withenhanced immunogenicity, and further, provides other related advantages.

SUMMARY OF THE INVENTION

Briefly stated, the present invention provides immunogenic syntheticfusion polypeptides which stimulate an immune response against Group Astreptococci. Within one aspect such polypeptides comprise (a) at leasttwo immunogenic polypeptides from a Group A streptococci of at least 10amino acids in length which are capable of stimulating an immuneresponse against Group A streptococci, and a peptide C terminal to theimmunogenic polypeptide which protects the immunogenicity of theimmunogenic portion. Within preferred embodiments, the C-terminalpeptide is not required to stimulate an immune response against Group Astreptococci and hence, may be an inconsequential non-immunogenicpeptide, or a reiterated immunogenic polypeptide. Within certainembodiments, the immunogenic polypeptide can be obtained from a widevariety of Group A streptococci (ranging from “1” to greater than “90”),including for example, Types 1, 1.1, 2, 3, 4, 5, 6, 11, 12, 13, 14, 18,19, 22, 24, 28, 30, 48, 49, 52, 55 and 56.

Within other aspects of the present invention, vaccinating agents areprovided for promoting an immune response against Group A streptococci,comprising (a) at least two immunogenic polypeptides from a Group Astreptococci of at least 10 amino acids in length which are capable ofstimulating a protective immune response against Group A streptococci,and (b) a peptide C terminal to the immunogenic polypeptide whichprotects the immunogenicity of the immunogenic portion, wherein theC-terminal peptide is not required to stimulate an immune responseagainst Group A streptococci. As above, the polypeptide may be selectedfrom a wide variety of Group A streptococci (ranging from “1” to greaterthan “90”), including for example, types 1.1, 2, 3, 4, 5, 6, 11, 12, 13,14, 18, 19, 22, 24, 28, 30, 48, 49, 52, 55 and 56. Within certainfurther embodiments, the vaccinating agent may further comprise anadjuvant, such as, for example, alum, Freund's adjuvant, and/or animmunomodulatory cofactor (e.g., IL-4, IL-10, γ-IFN, or IL-2, IL-12 orIL-15).

Also provided are methods for vaccinating a host against Group Astreptococci infections, comprising administering a vaccinating agent asdescribed above.

These and other aspects of the present invention will become evidentupon reference to the following detailed description and attacheddrawings. In addition, various references are set forth herein whichdescribe in more detail certain procedures or compositions (e.g.,plasmids, etc.), and are therefore incorporated by reference in theirentirety.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic of the hexavalent vaccine indicating the length ofeach emm gene fragment and the restriction sites that were synthesizedinto the original PCR primers. Each of the emm gene fragments starts atthe first codon that encodes the mature native protein except the emm3fragment, which represents codons 21-70.

FIG. 2 is a SDS-polyacrylamide gel electrophoresis of the purifiedhexavalent protein stained with Coomassie™ blue. Computer-assisted imageanalysis of the stained protein bands indicated that the hexavalentprotein (M.W. 45 kDa) accounted for 86% to 89% of the total protein ineach sample.

FIGS. 3A and 3B are ELISA's of antisera from three rabbits immunizedwith the hexavalent vaccine in either alum or CFA. Titers are expressedas the inverse of the last dilution of serum that resulted in an O.Dof >0.1. The ELISA antigen was the purified hexavalent protein. Eachsymbol represents serum from one rabbit.

FIGS. 4A-4F are ELISA's of antisera from rabbits immunized with thehexavalent protein in alum. Titers were determined using the purifiedpepsin-extracted M proteins (pep M) from the respective serotypes ofgroup A streptococci. Each symbol represents one of three immunizedrabbits.

FIG. 5 depicts in vitro opsonization assays of antisera from rabbitsimmunized with the hexavalent protein in alum. Rotation mixturesconsisted of the test organism, 0.1 ml of immune serum, and 0.4 ml ofnonimmune human blood. The mixture was rotated for 45 minutes and thepercentage of PMNs that had ingested or were associated withstreptococci was estimated by microscopic counts of stained smears. Ineach assay, the preimmune serum resulted in <10% percent opsonization.Each different bar represents serum from one of the three immunizedrabbits.

FIG. 6 is a graph which depicts opsonization of different strains withinthe same serotype of group A streptococci promoted by hexavalent rabbitantisera. Each symbol represents a strain of group A streptococci of theserotype indicated on the horizontal axis. Opsonization assays wereperformed as described below in the Examples.

FIGS. 7A and 7B show a nucleic acid sequence (SEQ ID NO:15) andpredicted amino acid sequence (SEQ ID NO: 16) of a hexavalent M proteinvaccine.

DETAILED DESCRIPTION OF THE INVENTION DEFINITIONS

Prior to setting forth the invention, it may be helpful to anunderstanding thereof to first set forth definitions of certain termsthat will be used hereinafter.

“Vaccinating Agent” refers to a composition which is capable ofstimulating a protective immune response within the host which receivesthe vaccinating agent. The vaccinating agent may be either protein, or,DNA-based (e.g., a gene delivery vehicle). Within further aspects, aprokaryotic host may be generated to be a vaccinating agent, anddesigned to express an immunogenic polypeptide or multivalent constructof the present invention (see, e.g., U.S. application Ser. No.07/540,586).

“Gene delivery vehicle” refers to a recombinant vehicle, such as arecombinant viral vector, a nucleic acid vector (such as plasmid), anaked nucleic acid molecule such as genes, a nucleic acid moleculecomplexed to a polycationic molecule capable of neutralizing thenegative charge on the nucleic acid molecule and condensing the nucleicacid molecule into a compact molecule, a nucleic acid associated with aliposome (Wang et al., PNAS 84: 7851, 1987), a bacterium, and certaineukaryotic cells such as a producer cell, that are capable of deliveringa nucleic acid molecule having one or more desirable properties to hostcells in an organism.

As noted above, the present invention provides vaccinating agentssuitable for preventing Group A streptococcal infections. Briefly, asdescribed in more detail below it has been discovered that, in order tooptimize the immunogenicity of all aspects of a multivalent vaccine.Within one aspect of the invention, immunogenic synthetic fusionpolypeptides which stimulate an immune response against Group Astreptococci are provided. Such polypeptides generally comprise (a) atleast two immunogenic polypeptides from a Group A streptococci of atleast 10 amino acids in length which are capable of stimulating animmune response against Group A streptococci, and (b) a peptide Cterminal to the immunogenic polypeptide which protects theimmunogenicity of the immunogenic portion, wherein the C-terminalpeptide is not required to stimulate an immune response against Group Astreptococci. Particularly preferred protective peptides are generallyat least ten amino acids in length, and may be 30 amino acids or longer.

Identification of Immunogenic Polypeptides, for Use in VaccinatingAgents

Immunogenic polypeptides suitable for use within the present inventionmay be readily identified and generated given the disclosure of thesubject application (see also Dale and Beachey, J. Exp. Med. 163:1191-1202; 1986; Beachey et al., Nature 292: 457-459, 1981; Dale et al.,J. Immunol. 151: 2188-2194; 1993; and U.S. Pat. Nos. 4,454,121;4,521,334; 4,597,967; 4,705,684; 4,919,930; and 5,124,153). Particularlypreferred polypeptides can be obtained within the 50 amino acid residuesof the N-terminus of an M protein.

Serotypes of Group A streptococci can be readily obtained from clinicalisolates, from university collections (e.g., Rockefeller UniversityCollection, 1230 York Avenue, New York, N.Y.) or from depositories suchas the American Type Culture Collection (10801 University Boulevard,Manassas, Va.). Furthermore, sequences for Group A streptococciserotypes are available from the Centers for Disease Control, Atlanta,Ga.

A. Identification of Opsonic Epitopes of M Proteins

To demonstrate directly that opsonic M protein epitopes could beseparated from autoimmune epitopes, peptides are copied from variousserotypes (e.g., the amino-terminal 20-50 amino acids of M5 (Beachey etal., “Purification and properties of M protein extracted from group Astreptococci with pepsin: Covalent structure of the amino terminalregion of the type 24 M antigen,” J. Exp. Med. 145: 1469-1483, 1977).SM5(1-20) failed to react with affinity purified pep M5 heart-reactiveantibodies (Beachey et al., “Purification and properties of M proteinextracted from group A streptococci with pepsin: Covalent structure ofthe amino terminal region of the type 24 M antigen,” J. Exp. Med 145:1469-1483, 1977). Rabbits immunized with SM5(1-20) coupled to tetanustoxoid developed high titers of antibodies against pep M5 that opsonizedtype 5 streptococci (Beachey et al., “Purification and properties of Mprotein extracted from group A streptococci with pepsin: Covalentstructure of the amino terminal region of the type 24 M antigen,” J.Exp. Med. 145: 1469-1483, 1977). Most importantly, none of the immunesera crossreacted with human myocardium.

B. Tissue-Crossreactive Epitopes of M Proteins

M proteins evoke antibodies that crossreact with a variety of humantissues and antigens within those tissues (Baird et al., “Epitopes ofgroup A streptococcal M protein shared with antigens of articularcartilage and synovium,” J. Immunol. 146: 3132-3137, 1991; Bronze, M. Sand Dale, J. B., “Epitopes of streptococcal M proteins that evokeantibodies that cross-react with human brain,” J. Immunol. 151:2820-2828., 1993; Dale, J. B and Beachey E. H., “Protective antigenicdeterminant of streptococcal M protein shared with sarcolemmal membraneprotein of human heart,” J. Exp. Med 156: 1165-1176, 1982). In order todetermine cross-reactivity, a series of overlapping peptides issynthesized that copies a selected fragment (e.g., M5), and used toeither inhibit or evoke tissue-crossreactive antibodies. For example,the myosin-crossreactive antibodies evoked by pep M5 in rabbits werealmost totally inhibited by peptide 84-116 of pep M5. This peptide spansthe region between the A and B repeats of M5 and includes the degenerateA6 repeat. Murine and human myosin-crossreactive antibodies reacted withan epitope in peptide 183-189, which is located in the region betweenthe B and C repeats of the intact M5 molecule.

Additional sarcolemmal membrane crossreactive epitopes are localized topeptide 164-197. Several epitopes of M5 that evoked antibodies thatcrossreacted with articular cartilage and synovium can also be foundwithin the B repeats and the region spanning the A and B repeats of M5.The brain-crossreactive epitopes of M6 that were shared with other Mproteins are localized to the B repeat region of the molecule.

Many of the tissue-crossreactive epitopes are shared among types 5, 6,18 and 19 M proteins (Bronze, M. S and Dale, J. B., “Epitopes ofstreptococcal M proteins that evoke antibodies that cross-react withhuman brain,” J. Immunol. 151: 2820-2828., 1993). Primary structuraldata reveals that all of these M proteins contain similar sequenceswithin their B repeats (Dale et al., “Recombinant tetravalent group Astreptococcal M protein vaccine,” J. Immunol. 151: 2188-2194, 1993; Daleet al., “Recombinant, octavalent group A streptococcal M proteinvaccine,” Vaccine 14: 944-948, 1996; Dale et al., “Type-specificimmunogenicity of a chemically synthesized peptide fragment of type 5streptococcal M protein,” J. Exp. Med. 158: 1727-1732, 1983), which ismost likely the location of the shared heart brain andjoint-crossreactive epitopes.

It should be emphasized that it is not necessary to localize thetissue-specific epitope, but rather, to first localize protectiveepitopes and ensure that they are not tissue-reactive.

Once a suitable immunogenic polypeptide for a selected serotype has beenidentified, it may be, optionally, combined with immunogenicpolypeptides from other serotypes, in order to construct a multivalentvaccine. In this regard, preferred vaccines include vaccines developedfrom a combination of serotypes such as 1, 1.1, 2, 3, 4, 5, 6, 11, 12,13, 14, 18, 19, 22, 24, 28, 30, 48, 49, 52 and 56 (for serotype 30 seeNakashima et al., Clinic Infec. Dis. 25: 260, 1997). Representativeexamples include vaccine such as 24, 5, 6, 19, 1, 3, λ; and 1, 3, 5, 6,18, 19, 22, 24, 28, 30, and λ, wherein X is the C-terminal protectivepolypeptide.

Preparation of Vaccinating Agents

Vaccinating agents of the present invention can be synthesizedchemically (see, e.g., Beachey et al., Nature 292: 457-459, 1981), orgenerated recombinantly. For recombinant production, PCR primers can besynthesized to amplify desired 5′ sequences of each emm gene, and eachprimer is extended to contain a unique restriction enzyme site used toligate the individual PCR products in tandem.

As noted above, the C-terminal portion of the vaccinating agent isconstructed so as to contain a selective portion that can be lost orcleaved in vivo without affecting the efficacy of the vaccine. This maybe accomplished by, for example, including an inconsequentialnon-immunogenic polypeptide at the end, or, including an immunogenicpolypeptide that does not adversely impact the efficiency of the vaccine(e.g., a reiterated immunogenic polypeptide may be included at the endof the vaccine). Furthermore, protective antigens from unrelatedpathogens can also be combined into a single polypeptide, which maycircumvent the need for carriers. Vaccines against some pathogens mightinclude T and B cell epitopes originally derived from different proteinson the same hybrid construct. Additionally, multivalent hybrid proteinsmay be sufficient conjugates in carbohydrate vaccines, such as those forS. pneumoniae, H influenza B or group B streptococci.

For protein expression, the multivalent genes are ligated into anysuitable replicating plasmid which is used to transform an appropriateprokaryote host strain. Prokaryotes include gram negative or grampositive organisms, for example E. coli or bacilli. Suitable prokaryotichosts cells for transformation include, for example, E. coli, Bacillussubtilis, Salmonella typhimurium, and various other species within thegenera Pseudomonas, Streptomyces, and Staphylococcus.

Expression vectors transfected into prokaryotic host cells generallycomprise one or more phenotypic selectable markers such as, for example,a gene encoding proteins that confer antibiotic resistance or thatsupplies an auxotrophic requirement, and an origin of replicationrecognized by the host to ensure amplification within the host. Otheruseful expression vectors for prokaryotic host cells include aselectable marker of bacterial origin derived from commerciallyavailable plasmids. This selectable marker can comprise genetic elementsof the cloning vector pBR322 (ATCC 37017). Briefly, pBR322 containsgenes for ampicillin and tetracycline resistance and thus providessimple means for identifying transformed cells. The pBR322 “backbone”sections are combined with an appropriate promoter and a mammalian ETFstructural gene sequence. Other commercially available vectors include,for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden), pQE30(His-tag expression vector), and pGEM1 (Promega Biotec, Madison, Wis.,USA).

Common promoter sequences for use within prokaryotic expression vectorsinclude β-lactamase (penicillinase), lactose promoter system (Chang etal., Nature 275: 615, 1978; and Goeddel et al., Nature 281: 544, 1979),tryptophan (trp) promoter system (Goeddel et al., Nucl. Acids Res. 8:4057, 1980; and EPA 36,776) and tac promoter (Sambrook et al., MolecularCloning: A Laboratorv Manual, Cold Spring Harbor Laboratory, (1989)). Aparticularly useful prokaryotic host cell expression system employs aphage λ P_(L) promoter and a c1857ts thermolabile repressor sequence.Plasmid vectors available from the American Type Culture Collection thatincorporate derivatives of the λ P_(L) promoter include plasmid pHUB2(resident in E. coli strain JMB9 (ATCC 37092)) and pPLc28 (resident inE. coli RR1 (ATCC 53082)).

Transformation of the host strains of E. coli is accomplished byelectroporation using standard methods (Dale et al., “Recombinanttetravalent group A streptococcal M protein vaccine,” J. Immunol. 151:2188-2194, 1993; Dale et al., “Recombinant, octavalent group Astreptococcal M protein vaccine,” Vaccine 14: 944-948, 1996). Successfultransformants are identified by colony blots using rabbit antiseraraised against one of the native M proteins or a synthetic peptide copyof the amino-terminus of one of the M proteins included in themultivalent protein.

The molecular size and antigenicity of the recombinant protein expressedby selected clones are determined by performing Western blots ofextracts of E. coli (Dale et al., “Recombinant tetravalent group Astreptococcal M protein vaccine,” J. Immunol. 151: 2188-2194, 1993)using rabbit antisera raised against each native M protein purified frompepsin extracts of live streptococci (Beachey et al., “Purification andproperties of M protein extracted from group A streptococci with pepsin:Covalent structure of the amino terminal region of the type 24 Mantigen,” J. Exp. Med. 145: 1469-1483, 1977). The multivalent gene issequenced by the dideoxy-nucleotide chain termination method to confirmthat each gene fragment is an exact copy of the native emm sequence.

Gene-delivery Vehicle-based Vaccines

Injection of mammals with gene delivery vehicles (e.g., naked DNA)encoding antigens of various pathogens has been shown to result inprotective immune responses (Ulmer et al., Science 259: 1745-9, 1993;Bourne et al., J. Infect. Dis. 173: 800-7, 1996; Hoffman et al., Vaccine12: 1529-33, 1994). Since the original description of in vivo expressionof foreign proteins from naked DNA injected into muscle tissue (Wolff etal., Science 247: 1465-8, 1990), there have been several advances in thedesign and delivery of DNA for purposes of vaccination.

The M protein vaccines described above are ideally suited for deliveryvia naked DNA because protective immunity is ultimately determined byantibodies. For example, within one embodiment the multivalent genes areligated into plasmids that are specifically engineered for mammaliancell expression (see, e.g., Hartikka et al., Hum Gene Ther 7: 1205-17,1996, which contains the promoter/enhancer element from cytomegalovirusearly gene, the signal peptide from human tissue plasminogen activatorand a terminator element from the bovine growth hormone gene). The Mprotein hybrid genes can be cloned into the plasmid which is used totransfect human cell lines to assure recombinant protein expression. Theplasmid is propagated in E. coli and purified in quantities sufficientfor immunization studies by cesium chloride gradient centrifugation.Mice are immunized with 50 ug of plasmid in 50 ul saline givenintramuscularly into the rectus femoris. Booster injections of the samedose are given at three and six weeks after the initial injection.

A wide variety of other gene delivery vehicles can likewise be utilizedwithin the context of the present invention, including for example,viruses, retrotransposons and cosmids. Representative examples includeadenoviral vectors (e.g., WO 94/26914, WO 93/9191; Yei et al., GeneTherapy 1: 192-200, 1994; Kolls et al., PNAS 91(1): 215-219, 1994;Kass-Eisler et al., PNAS 90(24): 11498-502, 1993; Guzman et al.,Circulation 88(6): 2838-48, 1993; Guzman et al., Cir. Res. 73(6):1202-1207, 1993; Zabner et al., Cell 75(2): 207-216, 1993; Li et al.,Hum Gene Ther. 4(4): 403-409, 1993; Caillaud et al., Eur. J. Neurosci.5(10): 1287-1291, 1993), adeno-associated type 1 (“AAV-1”) oradeno-associated type 2 (“AAV-2”) vectors (see WO 95/13365; Flotte etal., PNAS 90(22): 10613-10617, 1993), hepatitis delta vectors, live,attenuated delta viruses and herpes viral vectors (e.g., U.S. Pat. No.5,288,641), as well as vectors which are disclosed within U.S. Pat. No.5,166,320. Other representative vectors include retroviral vectors(e.g., EP 0 415 731; WO 90/07936; WO 91/02805; WO 94/03622; WO 93/25698;WO 93/25234; U.S. Pat. No. 5,219,740; WO 93/11230; WO 93/10218). Methodsof using such vectors in gene therapy are well known in the art, see,for example, Larrick, J. W and Burck, K. L., Gene Therapy: Applicationof Molecular Biology, Elsevier Science Publishing Co., Inc., New York,N.Y., 1991; and Kreigler, M., Gene Transfer and Expression. A LaboratoryManual, W. H. Freeman and Company, New York, 1990.

Gene-delivery vehicles may be introduced into a host cell utilizing avehicle, or by various physical methods. Representative examples of suchmethods include transformation using calcium phosphate precipitation(Dubensky et al., PNAS 81: 7529-7533, 1984), direct microinjection ofsuch nucleic acid molecules into intact target cells (Acsadi et al.,Nature 352: 815-818, 1991), and electroporation whereby cells suspendedin a conducting solution are subjected to an intense electric field inorder to transiently polarize the membrane, allowing entry of thenucleic acid molecules. Other procedures include the use of nucleic acidmolecules linked to an inactive adenovirus (Cotton et al., PNAS 89:6094, 1990), lipofection (Felgner et al., Proc. Natl. Acad. Sci. USA 84:7413-7417, 1989), microprojectile bombardment (Williams et al., PNAS 88:2726-2730, 1991), polycation compounds such as polylysine, receptorspecific ligands, liposomes entrapping the nucleic acid molecules,spheroplast fusion whereby E. coli containing the nucleic acid moleculesare stripped of their outer cell walls and fused to animal cells usingpolyethylene glycol, viral transduction, (Cline et al., Pharmac. Ther.29: 69, 1985; and Friedmann et al., Science 244: 1275, 1989), and DNAligand (Wu et al, J of Biol. Chem. 264: 16985-16987, 1989), as well aspsoralen inactivated viruses such as Sendai or Adenovirus.

Serum from mice immunized with gene delivery vehicles containingmultivalent M protein genes are assayed for total antibody titer byELISA using native M proteins as the antigen. Serum opsonic antibodiesare assayed as described above. Protective efficacy of DNA M proteinvaccines is determined by direct mouse protection tests using theserotypes of group A streptococci represented in the vaccine.

Formulation and Administration

For therapeutic use, vaccinating agents can be administered to a patientby a variety of routes, including for example, by intramuscular,subcutaneous, and mucosal routes. The vaccinating agent may beadministered as a single dosage, or in multiple units over an extendedperiod of time. Within preferred embodiments, the vaccinating agent isadministered to a human at a concentration of 50-300 ug per single siteintramuscular injection. Several injections can be given (e.g., three orfour) at least one month apart in order to further increase vaccineefficacy.

Typically, the vaccinating agent will be administered in the form of apharmaceutical composition comprising purified polypeptide inconjunction with physiologically acceptable carriers, excipients ordiluents. Such carriers will be nontoxic to patients at the dosages andconcentrations employed. Ordinarily, the preparation of suchcompositions entails combining the vaccinating agent with buffers,antioxidants such as ascorbic acid, low molecular weight (less thanabout 10 residues) polypeptides, proteins, amino acids, carbohydratesincluding glucose, sucrose or dextrans, chelating agents such as EDTA,glutathione and other stabilizers and excipients. Neutral bufferedsaline or saline mixed with conspecific serum albumin are exemplaryappropriate diluents.

Within preferred embodiments of the invention, the vaccinating agent iscombined with an adjuvant, such as, for example, Freund's adjuvant, alumand the like.

The following examples are offered by way of illustration, and not byway of limitation.

EXAMPLES Example 1 Construction and Expression of a Hexavalent FusionGene

A hexavalent emm gene was constructed using PCR to amplify specific 5′regions of the six different emm genes (24, 5, 6, 19, 1 and 3)essentially as described previously (Dale et al., “Recombinanttetravalent group A streptococcal M protein vaccine,” J. Immunol. 151:2188-2194, 1993; Dale et al., “Recombinant, octavalent group Astreptococcal M protein vaccine,” Vaccine 14: 944-948, 1996).

Briefly, the multivalent genes are constructed using PCR and primersthat specify specific 5′ emm gene fragments. The gene fragments mayrange in size from 30 bp to 300 bp. Chromosomal DNA from each serotypeof group A streptococcus is used as the template for the PCR reactions.For the hexavalent emm gene described in the example, the PCR primersare as follows: M24-1 TS (SEQ ID NO:1)             SphI 5′ GGG GGG GCATCG GTC GCG ACT AGG TCT CAG ACA GAT 3′ M24-1 BS (SEQ ID NO:2)            BamH1 5′ GGG GGG GGA TCC ACG TAG TTT CTC TTT AGC 3′ M5 TS(SEQ ID NO:3)            BamH1 5′ GGG GGG GGA TCC GCC GTG ACT AGG GGTACA 3′ M5 BS (SEQ ID NO:4)             SalI 5′ GGG GGG GTC GAC CTC AGTTTT TAA CCC TTC 3′ M6 TS (SEQ ID NO:5)             SalI 5′ GGG GGG GTCGAC AGA GTG TTT CCT AGG GGG 3′ M6 BS (SEQ ID NO:6)             NcoI 5′GGG GGG CCA TGG TAA CTT GTC ATT ATT AGC 3′ M19 TS (SEQ ID NO:7)                 NcoI 5′ GGG GGG CCA TGG AGA GTG CGT TAT ACT AGG 3′ M19BS (SEQ ID NO:8)                  PstI 5′ GGG GGG CTG CAG AGA TAA CTTCTC ATT CTG 3′ M1 TS (SEQ ID NO:9)             PstI 5′ GGG GGG CTG GAGAAC GGT GAT GGT AAT CCT 3′ M1 BS (SEQ ID NO:10)             KpnI 5′ GGGGGG GGT ACC AGC TCT CTT AAA ATC TCT 3′ M3 TS (SEQ ID NO:11)            KpnI 5′ GGG GGG GGT ACC TTG TTA GAT GAG GTT ACA 3′ M3 BS(SEQ ID NO:12)             ClaI 5′ GGG GGG ATC GAT ATT TAA CTC TTG TAACAG 3′ M24-2 TS (SEQ ID NO:13)              ClaI 5′ GGG GGG ATC GAT GTCGCG ACT AGG TCT CAG 3′ M24-2 BS (SEQ ID NO:14)             HindIII 5′GGG GGG AAG CTT TTA CTT ACG TGC CTC TAA TTC 3′

PCR is performed on the chromosomal template as previously described(Dale et al., “Recombinant tetravalent group A streptococcal M proteinvaccine,” J. Immunol. 151: 2188-2194, 1993). To assure ligation of thefragments in the correct orientation and reading frame, each PCR productis purified, ligated, and then subjected to PCR again using the forwardprimer from the 5′ fragment and the reverse primer from the 3′ fragment.For example, to construct a hexavalent emm gene containing DNA sequencesfrom types 24, 5, 6, 19, 1, and 3 M proteins, the M24 and M5 genefragments are amplified by PCR using the primers described above. ThePCR products are purified from agarose gels, cut with the appropriaterestriction enzyme, and ligated together (Dale et al., “Recombinanttetravalent group A streptococcal M protein vaccine,” J. Immunol. 151:2188-2194, 1993; Dale et al., “Recombinant, octavalent group Astreptococcal M protein vaccine,” Vaccine 14: 944-948, 1996). Theligation mixture is then amplified by PCR using the forward M24 primerand the reverse M5 primer. The resulting product of the appropriate sizeis then purified and ligated to the M6 and M19 gene fragment that wassimilarly constructed. After the final ligation reaction, the entiregene is amplified again by PCR, cut with the appropriate restrictionenzymes and ligated into a suitable expression vector. For the additionof the reiterated M24 gene fragment in the 3′ location, the plasmid waspurified from the host E. coli and a new PCR product from emm 24 wasforce cloned into the 3′ PstI restriction site.

The hexavalent gene was sequenced by the dideoxy-nucleotide chaintermination method to confirm that each gene fragment was an exact copyof the respective native emm sequence.

Example 2 Purification of a Hexavalent Vaccine

A. Purification

Transformed E. coli were grown in a shaking incubator to log phase in 11of LB containing 100 μg/ml ampicillin and 25 μg/ml kanamycin. IPTG (2mM) was added for the final four hours of growth. The cell pellet wassuspended in 30 ml PBS and lysed in a French pressure cell at 1000 psi.The hexavalent protein was purified from the supernatant using Ni-NTAresin according to the protocol provided by the manufacturer (Qiagen,Valencia, Calif.). The elution buffer containing the protein wasconcentrated from 15 ml to 5 ml in a spin filter (ULTRAFREE®-15,Millipore). Final purification was accomplished by gel filtration overSUPERDEX™ 75 (prep grade, Pharmacia Biotech). The active fraction wasidentified by Western blots (Dale, J. B and Beachey, E. H., “Multipleheart-cross-reactive epitopes of streptococcal M proteins,” J. Exp. Med.161: 113-122, 1985) using rabbit antiserum against pep M24 (Beachey etal., “Purification and properties of M protein extracted from group Astreptococci with pepsin: Covalent structure of the amino terminalregion of the type 24 M antigen,” J. Exp. Med. 145: 1469-1483, 1977).Total protein concentration was determined by standard methods and thesample was diluted in PBS to contain 200 μg/ml of hexavalent protein.Purity of the samples was determined by gel scanning (PHOTOSHOP™ digitalimage and COLLAGE™ image analysis).

B. Analysis of the Hexavalent Vaccine

The structure of the hybrid emm gene was confirmed by double-strandedsequencing methods after ligation into pQE30. The sequence of eachsubunit was identical to the respective native emm gene (FIG. 1). Thefragments were joined only by the two amino acids specified by eachunique restriction site used to facilitate their ligation (FIG. 1).

The purified hexavalent protein migrated on SDS-polyacrylamide gels withan apparent M. W of 45 kDa. (FIG. 2). Gel scan analysis revealed thatthe intact hexavalent protein accounted for approximately 90% of thetotal stainable protein in the gel. Western blots using antisera againstpep M24 showed that the majority of the remaining protein bands wereimmunoreactive and most likely were fragments of the hexavalent protein(data not shown).

Example 3 Immunization of Rabbits, and Testing of Antisera

A. Immunization

Two groups of three rabbits each were immunized with 100 μg ofhexavalent vaccine either precipitated with alum or emulsified incomplete Freund's adjuvant. For precipitation in alum, the hexavalentprotein (200 μg/ml) was added to an equal volume of aluminum hydroxide(2 mg/ml) (REHYDRAGEL™ HPA, Reheis, Inc., Berkeley Heights, N.J.) andmixed gently at 4° C. overnight. The hexavalent protein was alsoemulsified in CFA at a final concentration of 100 μg/ml. Rabbits thatreceived the hexavalent vaccine in alum were given 100 μg/ml as aninitial injection and the same dose was repeated at 4, and 8 weeks. Thesecond set of rabbits received 100 μg of hexavalent vaccine in CFAsubcutaneously as an initial injection and then booster injections ofthe same dose in saline were given at 4 and 8 weeks. Blood was obtainedprior to the first injection and at 2-week intervals thereafter.

Antibody assays. ELISAs were performed using purified nativepepsin-extracted M proteins (Beachey et al., “Purification andproperties of M protein extracted from group A streptococci with pepsin:Covalent structure of the amino terminal region of the type 24 Mantigen,” J. Exp. Med. 145: 1469-1483, 1977) or the purified hexavalentprotein, as previously described (Dale et al., “Heterogeneity oftype-specific and cross-reactive antigenic determinants within a singleM protein of group A streptococci,” J. Exp. Med 151: 1026-1038, 1980).Opsonic antibodies were detected by in vitro opsonization assays andindirect bactericidal assays (Beachey et al., “Human immune response toimmunization with a structurally defined polypeptide fragment ofstreptococcal M protein,” J. Exp. Med. 150: 862-877, 1979).

B. Detection of m Protein Antibodies.

The preimmune and immune animal sera are assayed by ELISA using thevaccine protein and the native pepsin-extracted M proteins assolid-phase antigens (Dale et al., “Heterogeneity of type-specific andcross-reactive antigenic determinants within a single M protein of groupA streptococci,” J. Exp. Med 151: 1026-1038, 1980). ELISA titers aredefined as the inverse of the last dilution of antisera resulting in anOD of >0.1 at 450 nm. The titers of immune sera against the native Mantigen are most likely to predict the levels of antibodies that areevoked by the recombinant protein that will react with the M protein onthe surface of the respective serotype of streptococcus (i.e. promoteopsonization).

C. Detection of Opsonic Antibodies.

Opsonic M protein antibodies correlate with protection against infectionwith the same serotype of group A streptococci (Lancefield, R. C.,“Current knowledge of the type specific M antigens of group Astreptococci,” J. Immunol. 89: 307-313, 1962; Lancefield, R. C.,“Persistence of type-specific antibodies in man following infection withgroup A streptococci,” J. Exp. Med. 110: 271-282, 1959). Two related invitro assays are used to detect opsonic antibodies in immune sera. Thefirst is a screening assay that measures opsonization in mixtures ofimmune serum, whole, nonimmune human blood and the test organism(Beachey et al., “Purification and properties of M protein extractedfrom group A streptococci with pepsin: Covalent structure of the aminoterminal region of the type 24 M antigen,” J. Exp. Med. 145: 1469-1483,1977). 0.1 ml of test serum is added to a standard number of bacteriaand incubated for 15 minutes at room temperature. 0.4 ml of lightlyheparinized human blood is added and the entire mixture is rotatedend-over-end at 37° C. for 45 minutes. At the end of the rotation,smears are prepared on microscope slides that are air-dried and stainedwith Wright's stain. “Percent opsonization” is quantitated by countingthe percentage of polymorphonuclear leukocytes that have ingested or areassociated with bacteria. An interpretable assay must have a preimmunecontrol value that is 10% opsonization or less.

Confirmation of the presence of opsonic antibodies is obtained byindirect bactericidal antibody assays according to the originaldescription by Lancefield (Lancefield, R. C., “Current knowledge of thetype specific M antigens of group A streptococci,” J. Immunol. 89:307-313, 1962). This assay is performed using test mixtures as describedabove except that fewer bacteria are added and the rotation is allowedto proceed for 3 hours. At the end of the rotation, pour plates are madein sheep blood agar and bacteria surviving are quantitated afterovernight growth at 37° C. Percent killing in the presence of immuneserum is calculated by comparing to the growth in nonimmune serum.

Example 4 Mouse Protection Assays

A. General Protocol

Protective efficacy of M protein vaccines is determined by eitherindirect or direct (passive or active immunization) mouse protectiontests. Indirect tests are performed by giving mice 1 ml of immune orpreimmune serum via the intraperitoneal (i.p.) route 24 hours prior tochallenge infections with the test organism given i.p. (Beachey et al.,“Human immune response to immunization with a structurally definedpolypeptide fragment of streptococcal M protein,” J. Exp. Med 150:862-877, 1979). For each test organism, groups of 25 mice receive eitherpreimmune or immune serum. The animals are then divided into 5 groups of5 mice each and 10-fold increasing challenge doses of virulentstreptococci are given to each subgroup. After 7 days of observation,the 50% lethal dose (LD₅₀) is calculated for each serotype tested.

Direct mouse protection tests are similarly performed except that miceare actively immunized with M protein vaccine prior to the challengeinfections. Each mouse receives 25-50 ug vaccine in alum givenintramuscularly (i.m.) at time 0, 4 weeks, and 8 weeks. Challengeinfections are performed ten weeks after the first injection. Controlanimals are sham immunized with alum alone. The LD50 is calculated andsignificance is determined using Fisher's exact test.

B. Protection

In order to show directly the protective efficacy of opsonic antibodiesevoked by the hexavalent vaccine, mice were immunized with the vaccineadsorbed to ALUM and then challenged with two of the serotypesrepresented in the vaccine. Female outbred white Swiss mice wereimmunized via the i.m. route in the hind leg according to the followingschedule: time 0, 25 μg; 3 weeks, 25 μg; 6 weeks, 50 μg; and 13 weeks,50 μg. Challenge experiments were performed on the 20 immunized mice and20 control, unimmunized mice (Table 1). The challenge strains were types24 and 19, with the reasoning that the M24 peptide is the largestfragment in the hexavalent protein and is reiterated and the M19fragment is one of two that are only 35 amino acids long. These twofragments should reflect the range of protective immunogenicity of thehexavalent protein. Intraperitoneal challenge of mice with virulentstreptococci is the most stringent laboratory assay for opsonicantibodies.

In this experiment, two groups of ten mice each were challenged with aninoculum that approximated the LD₇₀-LD₁₀₀ for each serotype, which was2×10⁴ CFU. The challenge experiments were begun 15 weeks after the firstdose of vaccine was administered and deaths were recorded for 10 days.The mice that were immunized with the hexavalent vaccine and challengedwith type 24 streptococci were significantly protected from deathcompared to the control group (p=0.0001). The mice challenged with type19 streptococci were protected by vaccination, but the level was notstatistically significant (p=0.15). Had the challenged group been twicethe size, the same level of protection would have resulted in astatistically significant survival rate. When the survival of the entireimmunized group of mice is analyzed, the level of protection was highlysignificant (p=0.0002). TABLE 1 Protective immunogenicity of thehexavalent vaccine in mice that were challenged i.p. with virulent type24 and type 19 streptococci #Dead/#Survived of Mice Challenged (%survival) Group Type 24 Type 19 Total Immunized mice 0/10 (100) 4/6 (60)4/16 (80) p = .0002* Control mice  9/1 (10) 7/3 (30) 16/4 (20)*p value was calculated using the Fisher exact test.

Example 5 Assays for Tissue-crossreactive Antibodies

To assure that none of the M protein vaccines evokestissue-crossreactive antibodies, indirect immunofluorescence assays areperformed using frozen sections of human heart, kidney, and brain (Dale,J. B and Beachey E. H., “Protective antigenic determinant ofstreptococcal M protein shared with sarcolemmal membrane protein ofhuman heart,” J. Exp. Med. 156: 1165-1176, 1982). Thin sections oftissue obtained at autopsy (4 um) are prepared on microscope slides andstored in a sealed box at −70° C. until use. Test serum is diluted 1:5in PBS and dropped onto the tissue section. Control slides are made withpreimmune serum and PBS. The slides are incubated at ambient temperaturefor 30 minutes and then washed three times in PBS in a slide holder.Fluorescein-labeled goat anti-IgG/IgM/IgA is diluted 1:40 in PBS anddropped onto the slides which are again washed, dried, and mounted with1% Gelvetol and a coverslip. Fluorescence is detected using a ZeissAxiophot microscope equipped with a xenon light source.Immunofluorescence is recorded using a scale of 0-4+, with 0 being nofluorescence and 4+being that obtained with a standard, positiveantiserum raised in rabbits against whole type 5 M protein (Dale, J. Band Beachey, E. H., “Multiple heart-cross-reactive epitopes ofstreptococcal M proteins,” J. Exp. Med 161: 113-122, 1985).

Example 6 Comparison of the Immunogenicity of a Hexavalent vaccineDelivered in Alum Versus Freund's Adjuvant

Three rabbits each were immunized with 100 μg doses of the hexavalentvaccine in either alum or CFA. Booster injections of the same dose weregiven at 4 and 8 weeks in either alum or saline, respectively. ELISAtiters were determined using the purified hexavalent protein as thesolid phase antigen (FIG. 3). Sera from the animals that received thehexavalent vaccine in alum had antibody titers that were equal to orgreater than the sera from rabbits that received the same dose in CFA.In a subsequent experiment, three rabbits were immunized i.m. with 100μg of the hexavalent vaccine in saline alone according to the sameschedule. None of these rabbits developed significant antibody titersagainst either the immunogen or the respective pep M proteins (data notshown). These data indicate that alum is a suitable and necessaryadjuvant for the multivalent vaccine and is equal to the adjuvantactivity of CFA in combination with the hexavalent protein.

Example 7 Protective Immunogenicity of the Component Subunits of aHexavalent Vaccine

One of the major goals of this study was to design a multivalent, hybridprotein that retained the immunogenic properties of each M proteinsubunit. ELISAs were performed on sera obtained from the three rabbitsimmunized with the hexavalent vaccine in alum (FIG. 4). In each case theELISA antigen was the purified pepsin-extracted M protein. Thus, theassay measures only the antibodies evoked by the hexavalent protein thatreact with the native M protein and not the antibodies that may bespecific for the joining segments or conformations that are not presentin the native M proteins. The hexavalent protein evoked significantlevels of antibodies against each M protein represented in the vaccineconstruct (FIG. 4). Importantly, none of the antisera containedantibodies that crossreacted with human heart tissue or kidney tissue,as determined by indirect immunofluorescence assays (data not shown).

Sera from all three rabbits contained significant levels of opsonicantibodies against each serotype of group A streptococci represented inthe vaccine (FIG. 5). These results were confirmed by indirectbactericidal assays using one of the immune sera (Table 2). Takentogether, the results indicate that the individual components of thehexavalent vaccine retain the conformation and immunogenicity necessaryto elicit antibodies that react with the native M proteins on thesurface of each respective serotype of group A streptococci.

Table 2. Indirect bactericidal assay of rabbit antiserum against thehexavalent M protein vaccine. CFU Surviving 3 hr rotation: PercentSerotype Inoculum(CFU) Preimmune Immune Reduction 24 12 2890 0 100 5 113260 0 100 6 6 2640 0 100 19 6 1580 0 100 1 8 2670 490 82 3 11 1720 1099

From the foregoing it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

1. A method for eliciting an immune response against Group A streptococci, comprising administering to a patient a recombinant fusion polypeptide wherein said recombinant fusion polypeptide comprises a multivalent immunogenic portion fused to an immunogenic polypeptide carboxy-terminal to the multivalent immunogenic portion, wherein the multivalent immunogenic portion comprises at least two immunogenic amino-terminal polypeptides of Group A streptococcal M protein from at least two different Group A streptococcal serotypes, and wherein the immunogenic polypeptide carboxy-terminal to the multivalent immunogenic portion is a reiteration of the immunogenic amino-terminal polypeptide from the amino terminus of the multivalent immunogenic portion, thereby eliciting an immune response against Group A streptococci.
 2. The method according to claim 1 wherein at least one of said immunogenic amino-terminal polypeptides of the fusion polypeptide is from a Group A streptococcal serotype selected from the group consisting of 1, 2, 3, 4, 5, 6, 11, 12, 13, 14, 18, 19, 22, 24, 28, 30, 48, 49, 52, and
 56. 3. The method according to claim 1 wherein the multivalent immunogenic portion of the fusion polypeptide consists of six immunogenic amino-terminal polypeptides of Group A streptococcal M protein from six different Group A streptococcal serotypes.
 4. The method according to claim 3 wherein the six different Group A streptococcal serotypes are 1, 3, 5, 6, 19, and
 24. 5. The method according to claim 1 wherein the multivalent immunogenic portion of the fusion polypeptide consists of ten immunogenic amino-terminal polypeptides of Group A streptococcal M protein from ten different Group A streptococcal serotypes.
 6. The method according to claim 5 wherein the ten different Group A streptococcal serotypes are 1, 3, 5, 6, 18, 19, 22, 24, 28, and
 30. 7. The method according to any one of claims 1 to 3 wherein at least one of the immunogenic amino-terminal polypeptides of the fusion polypeptide is from a Group A streptococcal serotype
 1. 8. The method according to any one of claims 1 to 3 wherein at least one of the immunogenic amino-terminal polypeptides of the fusion polypeptide is from a Group A streptococcal serotype
 2. 9. The method according to any one of claims 1 to 3 wherein at least one of the immunogenic amino-terminal polypeptides of the fusion polypeptide is from a Group A streptococcal serotype
 11. 10. The method according to any one of claims 1 to 3 wherein at least one of the immunogenic amino-terminal polypeptides of the fusion polypeptide is from a Group A streptococcal serotype
 13. 11. The method according to any one of claims 1 to 3 wherein at least one of the immunogenic amino-terminal polypeptides of the fusion polypeptide is from a Group A streptococcal serotype
 19. 12. The method according to any one of claims 1 to 3 wherein at least one of the immunogenic amino-terminal polypeptides of the fusion polypeptide is from a Group A streptococcal serotype
 22. 13. The method according to any one of claims 1 to 3 wherein at least one of the immunogenic amino-terminal polypeptides of the fusion polypeptide is from a Group A streptococcal serotype
 28. 14. The method according to any one of claims 1 to 3 wherein the administered fusion polypeptide elicits an immune response comprising opsonic antibodies against Group A streptococcal M protein that do not cross-react with human tissue.
 15. The method according to claim 1 wherein the recombinant fusion polypeptide further comprises a marker encoded by an expression vector.
 16. The method according to claim 15 wherein the expression vector is a His-tag vector.
 17. The method according to claim 16 wherein the marker binds to nickel resin.
 18. The method according to any one of claims 1 to 3 wherein the immunogenic polypeptides of the fusion polypeptide are joined by amino acids specified by a restriction enzyme site.
 19. The method according to claim 1 wherein the patient is human.
 20. The method according to claim 1 or claim 19 wherein the recombinant fusion polypeptide is administered via a subcutaneous route, an intramuscular route, or a mucosal route.
 21. The method according to claim 20 wherein the recombinant fusion polypeptide is administered via an intramuscular route to a human at a concentration of 50 μg to 300 μg.
 22. The method according to any one of claims 1 to 3 wherein the recombinant fusion polypeptide is further formulated with an adjuvant.
 23. The method according to claim 22 wherein the adjuvant is alum.
 24. The method according to claim 22 wherein the recombinant fusion polypeptide is further formulated with an immunomodulatory cofactor.
 25. A method for eliciting an immune response against Group A streptococci, comprising administering to a patient a pharmaceutical composition comprising a recombinant fusion polypeptide according to claim 1, and a pharmaceutically acceptable excipient, carrier, stabilizer or diluent, thereby eliciting an immune response against Group A streptococci.
 26. The method according to claim 25 wherein the composition further comprises an adjuvant.
 27. The method according to claim 26 wherein the adjuvant is alum.
 28. The method according to claim 25 or claim 26 wherein the pharmaceutically acceptable excipient, carrier, stabilizer or diluent comprises at least one of a buffer, antioxidant, carbohydrate, and chelating agent.
 29. The method according to claim 25 or claim 26 wherein the composition further comprises an immunomodulatory cofactor.
 30. The method according to claim 29 wherein the immunomodulatory cofactor is selected from the group consisting of IL-4, IL-10, γ-IFN, IL-2, IL-12, and IL-15.
 31. The method according to claim 25 wherein at least one of said immunogenic amino-terminal polypeptides of the fusion polypeptide is from a Group A streptococcal serotype selected from the group consisting of 1, 2, 3, 4, 5, 6, 11, 12, 13, 14, 18, 19, 22, 24, 28, 30, 48, 49, 52, and
 56. 32. The method according to claim 25 wherein the multivalent immunogenic portion of the fusion polypeptide consists of six immunogenic amino-terminal polypeptides of Group A streptococcal M protein from six different Group A streptococcal serotypes.
 33. The method according to claim 32 wherein the six different Group A streptococcal serotypes are 1, 3, 5, 6, 19, and
 24. 34. The method according to claim 25 wherein the multivalent immunogenic portion of the fusion polypeptide consists of ten immunogenic amino-terminal polypeptides of Group A streptococcal M protein from ten different Group A streptococcal serotypes.
 35. The method according to claim 34 wherein the ten different Group A streptococcal serotypes are 1, 3, 5, 6, 18, 19, 22, 24, 28, and
 30. 36. The method according to claim 25 or claim 26 wherein at least one of the immunogenic amino-terminal polypeptides of the fusion polypeptide is from a Group A streptococcal serotype
 1. 37. The method according to claim 25 or claim 26 wherein at least one of the immunogenic amino-terminal polypeptides of the fusion polypeptide is from a Group A streptococcal serotype
 2. 38. The method according to claim 25 or claim 26 wherein at least one of the immunogenic amino-terminal polypeptides of the fusion polypeptide is from a Group A streptococcal serotype
 11. 39. The method according to claim 25 or claim 26 wherein at least one of the immunogenic amino-terminal polypeptides of the fusion polypeptide is from a Group A streptococcal serotype
 13. 40. The method according to claim 25 or claim 26 wherein at least one of the immunogenic amino-terminal polypeptides of the fusion polypeptide is from a Group A streptococcal serotype
 19. 41. The method according to claim 25 or claim 26 wherein at least one of the immunogenic amino-terminal polypeptides of the fusion polypeptide is from a Group A streptococcal serotype
 22. 42. The method according to claim 25 or claim 26 wherein at least one of the immunogenic amino-terminal polypeptides of the fusion polypeptide is from a Group A streptococcal serotype
 28. 43. The method according to claim 25 or claim 26 wherein the administered composition elicits an immune response comprising opsonic antibodies against Group A streptococcal M protein that do not cross-react with human tissue.
 44. The method according to claim 25 or claim 26 wherein the recombinant fusion polypeptide further comprises a marker encoded by an expression vector.
 45. The method according to claim 44 wherein the expression vector is a His-tag vector.
 46. The method according to claim 45 wherein the marker binds to nickel resin.
 47. The method according to claim 25 or claim 26 wherein the immunogenic polypeptides of the fusion polypeptide are joined by amino acids specified by a restriction enzyme site.
 48. The method according to claim 25 wherein the patient is human.
 49. The method according to claim 25 or claim 48 wherein the composition is administered via a subcutaneous route, an intramuscular route, or a mucosal route.
 50. The method according to claim 49 wherein the composition is administered via an intramuscular route to a human at a concentration of 50 μg to 300 μg.
 51. The method according to claim 1 or claim 25 wherein the elicited immune response is a protective immune response. 